This invention is in the field of molecular biology, and involves methods and compositions for regulating unwanted cell growth through the regulation of the activity of certain guanine nucleotide exchange factors.
Ras is a member of a superfamily of GTPases that regulate diverse signaling pathways. Ras itself has been shown to be involved in regulating cell growth and differentiation (See, Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654). A subfamily of Ras consists of Rho, Rac, and Cdc42. These GTPase have also been shown to be involved in regulating cell growth, particularly as relating to cellular transformation, as well as controlling the formation of focal contacts and alterations in the actin cytoskeleton which occur upon growth factor stimulation (See, Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T. and Gutkind, J. S. (1995) Cell 81, 1137-1146; Hill, C. S., Wynne, J. and Treisman, R. (1995) Cell 81, 1159-1170; Kozma, R., Ahmed, S., Best, A. and Lim, L. (1995) Mol. Cell. Biol. 15, 1942-1952; Minden, A., Lin, A., Claret, F.-X., Abo, A. and Karin, M. (1995) Cell 81, 1147-1157; Nobes, C. D. and Hall, A. (1995) Cell 81, 53-62; Olson, M. F., Ashworth, A. and Hall, A. (1995) Science 269, 1270-1272). Common to all Ras family members is their ability to cycle between inactive (GDP bound) and active (GTP bound) states. In this regard, these GTPases act as molecular switches, capable of processing information and then disseminating that information to control a specific pathway.
This property of cycling between GTP and GDP states has provided a means to identify and purify proteins which regulate the nucleotide state of Ras and Ras related GTPases. See, Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654.
By monitoring the hydrolysis of GTP to GDP, GTPase activating proteins (GAPs) have been characterized for many members of the Ras family. See, Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654; Barfod, E. T., Zheng, Y., Kuang, W.-J., Hart, M. J., Evans, T., Cerione, R. A. and Ashkenaz, A. (1993) J. Biol. Chem. 268, 26059-26062; Lamarche, N. and Hall, A. (1994) Trends Genet. 10, 436-440; Cerione, R. A. and Zheng, Y. (1996) Current Opinion in Cell Biology 8, 216-222. The latter reference provides a good discussion of the properties of those proteins that affect the guanine nucleotide state of Ras and Ras related proteins. Guanine nucleotide dissociation inhibitors (GDIs) were identified based on their ability to inhibit the dissociation of GDP. It has subsequently been determined that they also bind to the GTP state, inhibiting the intrinsic and GAP stimulated GTP hydrolysis. See, Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654. In general, GAPs and effectors have a high affinity for the GTP-bound state, while GDI proteins bind most tightly to the GDP-bound state. These properties have been exploited to purify effectors for Cdc42Hs (See, Bagrodia, S., Taylor, S. J., Creasy, C. L., Chernoff, J. and Cerione, R. A. (1995) J. Biol. Chem. 270, 22731-22737; Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-s. and Lim, L. (1994)Nature 367, 40-46; Martin, G. A., Bollag, G., McCormick, F. and Abo, A. (1995) EMBO J. 14, 1970-1978), Ras (See, Moodie, S. A., Willumsen, B. M., Weber, M. J. and Wolfman, A. (1993) Science 260, 1658-1661; Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D. and Downward, J. (1994) Nature 370, 527-532) and Rho (See, Leung, T., Manser, E., Tan, L. and Lim, L. (1995) J. Biol. Chem. 270, 29051-29054; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuki, A. and Narumiya, S. (1996) Science 271, 645-648). An affinity approach has also been employed with Cdc42Hs-GTP and has led to the characterization of IQGAP 1, a potential mediator for observed cytoskeletal events induced by Cdc42. See, Hart, M. J., Callow, M., Souza, B. and Polakis, P. (1996) EMBO J. 15, 2997-3005.
A modification of this affinity approach can also be used to identify and purify guanine nucleotide exchange factors (GEFs). GEFs can be distinguished from other regulatory proteins by their ability to interact preferentially with the nucleotide-depleted state of G-proteins. See, Hart, M. J., Eva, A., Zangrilli, D., Aaronson, S. A., Evans, T., Cerione, R. A. and Zheng, Y. (1994) J. Biol. Chem. 269, 62-65; Mosteller, R. D., Han, J. and Broek, D. (1994) Mol. Cell. Biol. 14, 1104-1112. By stimulating the dissociation of GDP and subsequent binding of GTP, GEFs play an important role in the activation of Ras-like proteins. For example, Ras is converted to its GTP-bound form by the growth-factor stimulated translocation of Sos, a Ras-specific GEF. See, Buday, L. and Downward, J. (1993)Cell 73, 611-620.
The characterization of GEFs that specifically activate Rac family members will help elucidate signalling pathways in which these GTPases participate, and thus lead to a better understanding of the molecular basis of cell growth. This, in turn, will enable the identification of drugs for preventing or treating diseases where uncontrolled cell growth is the cause. Because Rac plays a key role in signal transduction and cell growth, the identification and properties of Rac GEFs is presently receiving considerable scientific attention. One such Rac GEF is known, Tiam-1. See, Michiels, F., Habets, G. G., Stam, J. C., van der Kammen, R. A., and Collard, J. G. (1995) Nature 375, 338-340. See also, Eva, A. and Aaronson, S. A. (1985) Nature 316, 273-275; Toksoz, D. and Williams, D. A. (1994) Oncogene 9, 621-628.
The present invention relates to all aspects of a guanine exchange factor (GEF), in particular, a Rac-GEF. A GEF modulates cell signaling pathways, both in vitro and in vivo, by modulating the activity of a GTPase. By way of illustration, a Rac-GEF, which modulates the activity of a Rac GTPase, is described. However, the present invention relates to other GEFs, especially other Rac-GEFs.
The present invention preferably relates to an isolated Rac-GEF polypeptide characterized by having a Src homology, Dbl homology and pleckstrin homology domains, and variants thereof, or fragments of such polypeptides, nucleic acids coding for such Rac-GEFs or nucleic acid fragments, and derivatives of the polypeptides and nucleic acids.
The invention also relates to methods of using such polypeptides, nucleic acids, or derivatives thereof, e.g., in therapeutics, diagnostics, and as research tools.
Another aspect of the present invention involves antibodies and other ligands which recognize the invention Rac-GEF, regulators of Rac-GEF activity and other GEFs, and methods of treating pathological conditions associated or related to such Rac GTPase.
The invention also relates to methods of testing for and/or identifying agents which regulate GEF by measuring their effect on GEF activity, e.g., in binding to a GTPase and/or nucleotide exchange activity.
The invention also relates to methods of assaying for GEF activity, preferrably using activators of GEF activity.
These and other aspects of the invention will become apparent upon a full considertion of the following disclosure.